RNA content in mammalian muscle fibres

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This important research was part of the PhD work carried out by Petra Habets in the research group headed by Professor Anthony J Sargeant. It was a collaboration and jointly supervised by Anton Moorman of the Academic Medical Centre, of the University of Amsterdam. Sadly one of the inspirations for this research and close friend Jose Sant’Ana Pereira died, much too young, a few years after this work was published while working in the University of Madison, Wisconsin.

RNA content differs in slow and fast muscle fibers: implications for interpretation of changes in muscle gene expression

Petra E.M.H. Habets, Diego Franco, Jan M. Ruijter, Anthony J. Sargeant, José A.A. Sant’Ana Pereira, Anton F.M. Moorman.

Journal of Histochemistry and Cytochemistry
J Histochem Cytochem. 1999 Aug;47(8):995-100
Quantification of a specific muscle mRNA per total RNA (e.g., by Northern blot analysis) plays a crucial role in assessment of developmental, experimental, or pathological changes in gene expression. However, total RNA content per gram of a particular fiber type may differ as well.
We have tested this possibility in the distinct fiber types of adult rat skeletal muscle.
Sections of single fibers were hybridized against 28S rRNA as a marker for RNA content.
Quantification of the hybridization showed that the 28S rRNA content decreases in the order I>IIA>IIX>IIB, where Type I fibers show a five- to sixfold higher expression level compared to Type IIB fibers. Results were verified with an independent biochemical determination of total RNA content performed on pools of histochemically defined freeze-dried single fibers. In addition, the proportion of myosin heavy chain (MHC) mRNA per microgram of total RNA was similar in slow and fast fibers, as demonstrated by Northern blot analysis.
Consequently, Type I fibers contain five- to sixfold more MHC mRNA per microgram of tissue than IIB fibers. These differences are not reflected in the total fiber protein content.
This study implies that proper assessment of mRNA levels in skeletal muscle requires evaluation of total RNA levels according to fiber type composition
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Human Muscle Fibre Types

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In this important series of studies a collaboration between the research group in Amsterdam led by Anthony Sargeant and that in London under the direction of Professor Geoffrey Goldspink used new techniques based on microdissection of fragments of human muscle fibre obtained by needle biopsy.
Characterization of human skeletal muscle fibres according to the myosin heavy chains they express

Steven EnnionJose A A Sant’ana PereiraAnthony J SargeantArchie YoungGeoffrey Goldspink.

Journal of Muscle Research and Cell Motility
J Muscle Res Cell Motil. 1995 Feb;16(1):35-43
Using a method of single muscle fibre analysis, we investigated the presence of RNA transcripts for various isoforms of the myosin heavy chain (MyoHC) gene in histochemically, immunohistochemically and electrophoretically characterized individual muscle fibres (n = 65) from adult human vastus lateralis muscle. A cDNA clone isolated in this study was shown to contain the 3′ end of a previously uncharacterized human MyoHC gene which is expressed specifically in human fast IIA muscle fibres and we conclude that this clone contains part of the human fast IIA MyoHC gene. In all the fibres histochemically, immunohistochemically and electrophoretically characterized as containing the previously classified IIB MyoHC (n = 23), it was shown that the human equivalent to the rat type IIX MyoHC gene is expressed. This observation was taken to suggest that the previously classified IIB muscles fibres in human muscle express a MyoHC isoform equivalent to the rat IIX, not the IIB, and would therefore be more accurately classified as IIX fibres.

Research into RNA in skeletal muscle

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Petra Habets was a PhD student directed by Anthony Sargeant. This research publication  was part of her PhD thesis. It was a collaborative research project in Amsterdam that developed out of research first pursued by Jose Sant’ana Periera, a previous PhD student in Tony Sargeant’s department.
Journal of Histochemistry and Cytochemistry
J Histochem Cytochem. 1999 Aug;47(8):995-1004

Quantification of a specific muscle mRNA per total RNA (e.g., by Northern blot analysis) plays a crucial role in assessment of developmental, experimental, or pathological changes in gene expression.

However, total RNA content per gram of a particular fiber type may differ as well. We have tested this possibility in the distinct fiber types of adult rat skeletal muscle. Sections of single fibers were hybridized against 28S rRNA as a marker for RNA content. Quantification of the hybridization showed that the 28S rRNA content decreases in the order I>IIA>IIX>IIB, where Type I fibers show a five- to sixfold higher expression level compared to Type IIB fibers. Results were verified with an independent biochemical determination of total RNA content performed on pools of histochemically defined freeze-dried single fibers. In addition, the proportion of myosin heavy chain (MHC) mRNA per microgram of total RNA was similar in slow and fast fibers, as demonstrated by Northern blot analysis. Consequently, Type I fibers contain five- to sixfold more MHC mRNA per microgram of tissue than IIB fibers. These differences are not reflected in the total fiber protein content. This study implies that proper assessment of mRNA levels in skeletal muscle requires evaluation of total RNA levels according to fiber type composition

RNA content varies in different types of mammalian muscle fibres

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Research carried out in Amsterdam under the joint supervision of Professors Anton F.M. Moorman and Anthony J Sargeant by their highly talented PhD student Petra Habets.

Quantification of a specific muscle mRNA per total RNA (e.g., by Northern blot analysis) plays a crucial role in assessment of developmental, experimental, or pathological changes in gene expression.

However, total RNA content per gram of a particular fiber type may differ as well. We have tested this possibility in the distinct fiber types of adult rat skeletal muscle. Sections of single fibers were hybridized against 28S rRNA as a marker for RNA content. Quantification of the hybridization showed that the 28S rRNA content decreases in the order I>IIA>IIX>IIB, where Type I fibers show a five- to sixfold higher expression level compared to Type IIB fibers. Results were verified with an independent biochemical determination of total RNA content performed on pools of histochemically defined freeze-dried single fibers. In addition, the proportion of myosin heavy chain (MHC) mRNA per microgram of total RNA was similar in slow and fast fibers, as demonstrated by Northern blot analysis. Consequently, Type I fibers contain five- to sixfold more MHC mRNA per microgram of tissue than IIB fibers. These differences are not reflected in the total fiber protein content. This study implies that proper assessment of mRNA levels in skeletal muscle requires evaluation of total RNA levels according to fiber type composition

http://www.pubfacts.com/author/Anthony+J+Sargeant